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Plant and Cell Physiology, 1986, Vol. 27, No. 3 431-441
© 1986

Article

In Vitro Synthesis of Pteroylpoly-{gamma}-glutamates by Cotyledon Extracts of Pisum sativum L.

Patrick Y. Chan, John W. Coffin and Edwin A. Cossins

Department of Botany, University of Alberta Edmonton, Alberta, Canada, T6G 2E9

The formation of folylpolyglutamate derivatives by germinating pea seeds (Pisum sativum L. cv Homesteader) was examined in vivo and in vitro. Differential microbiological assay of cotyledon extracts showed that total folate concentrations increased from 163 ng folate equivalents per g fresh weight at day 1 to 680 ng per g fresh weight at day 3 of germination. Over a 7 day germination period, folylpolyglutamate derivatives accounted for 46–73% of the total cotyledonary folate pool. The concentration of these polyglutamate forms of folate increased 6.5 fold during the first four days of germination and then remained relatively constant.

Dialyzed extracts of 1–4 day old cotyledons had ability to incorporate [3H]glutamate and [14C]tetrahydrofolate into folylpolyglutamates. This activity was mainly associated with protein precipitating at 35–45% of saturation with ammonium sulphate. The folylpolyglutamate synthetase of pea cotyledons had requirements for ATP and the monoglutamate of tetrahydrofolate. The latter folate was a more effective substrate than 5,10-methylenetetrahydrofolate but the diglutamate of unsubstituted tetrahydrofolate was not utilized. Ion exchange chromatography of the reaction products suggested that [3H]glutamate and [14C]tetrahydrofolate were incorporated into di-, and tetraglutamates of tetrahydrofolate. Folates of longer glutamyl chain lengths were only detected when the synthetase reaction proceeded for 12 h or longer.

(Received August 23, 1985; Accepted January 22, 1986)
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